Quantitative determination of unknown substances contained in biological materials and foods utilizing an enzymatic reaction has been widely practiced since there are various advantages for the enzymatic reaction that it is high speed in response and it has a substrate specificity.
And in recent years, the public attention has been focused on enzymatic quantitative determination methods using an enzyme electrode not only in the medical field but also in the food and drug fields since there are advantages that the use of an enzyme in a small amount makes it possible to practice microanalysis of a trace substance contained in a biological material, food, drug or the like. In addition to this, the enzyme can be repeatedly used.
For instance, among such enzymatic quantitative determination methods, there is known the so-called amperometric method of measuring an electric current in the electrode reaction of a material produced or consumed by an enzymatic reaction by applying a constant voltage on the enzymatic electrode. And, the amperometric method has been generally evaluated as being the most preferred since there are various advantages that the constitution of the electrodes to be used is simple, the electrode may be easily made highly sensitive, and in the case of the electrode utilizing the enzymatic reaction of an oxidase producing hydrogen peroxide, it is excellent in response speed and sensitivity.
However, there still remain an unsolved problem for said amperometic method that in the case where a sample to be examined contains a reducing substance, such reducing substance often renders it incapable of precisely determining the content of an objective substance to be observed.
For instance, the mechanism of quantitatively determining the glucose content in a sample using glucose-oxidase (GOD) is based on the following reactions: ##STR1## wherein hydrogen peroxide produced in the reaction (i) will cause an electrode reaction of the above reaction formula (ii) with the application of a voltage of about +0.6 V .nu.s a saturated calomel electrode (hereinafter referred to as "SCE") where an electric current which is caused at that time is relative to the glucose content in the sample. Because of this, the quantitative determination of the glucose content becomes possible.
In this system, there is a high substrate specificity against the glucose to be determined in the enzymatic reaction (i). However, in the electrode reaction (ii), various reducing materials, i.e., interfering materials contained in the sample, are oxidized on the electrode to cause an extra electric current, which will be measured together with the electric current based on the glucose. Because of this, it becomes impossible to obtain an accurate value for the glucose content. As such interfering material present in foods or biological fluids, there are known ascorbic acid, urea, reduced glutathione, thyrosin, etc.
In order to solve the above problem, there has been made a proposal of providing a permselective membrane consisting of acetylcellulose or the like between the enzyme immobilized layer and the electroconductive substrate made of platinum, etc. in the system of measuring the amount of hydrogen peroxide produced as a result of the oxidase reaction in a way that the measurement can be carried out under the condition of allowing the selective permeation of the hydrogen peroxide.
Even for this proposal, there is a problem that since there exists a clearance filled with an electrolyte solution, buffer solution or the like between the permselective membrane - enzyme layer complex system and the electroconductive substrate, it takes some period of time for the resulting hydrogen peroxide to be diffused in the permselective membrane and the clearance, and because of this, the response speed will be delayed. In addition to this, there is another problem that because the resulting hydrogen peroxide is diluted within the clearance, it is necessitated to highten the sensitivity of the electric detecting circuits.
There has been made an attempt to make the permselective membrane thinner in order to eliminate the above problems. However, such attempt is not effective because the foregoing interfering materials partly become permeated in this case.
There has been made a further attempt to make the scale of the electrode smaller, that is, to be a microelectrode, in order to prevent the response speed from being delayed due to the foregoing diffusion or/and the foregoing dilution of the resulting hydrogen peroxide.
However this attempt is not practical because there are problems in the case of such microelectrode that the current density will become smaller as the electrode surface area diminishes in size and as a result, the electric circuit becomes necessary to have a high sensitivity.
There has been made a still further attempt to dispose the foregoing permselective membrane directly on the surface of the electroconductive substrate by means of the casting technique. However, for the electrode system prepared under this attempt, there is a problem that the adhesion between the foregoing permselective membrane and the electroconductive substrate is not strong enough and its durability is not satisfactory. This problem becomes significant especially in the case of developing an automatic and high speed flow-type enzyme-based analyzer since there will be given a specific shearing force based on the liquid flow onto the permselective membrane.